: The molecular basis for the cellular actions of insulin and related hormones/growth factors is incompletely understood. The insulin receptor is a ligand-activated, tyrosine-specific protein kinase, and much evidence now indicates that the activation of the receptor tyrosine phosphotransferase function is the first intracellular step in insulin action. The identity of the succeeding steps, which couple the activated receptor kinase to the rapid activation of intracellular enzymes and transport systems, as well as changes in gene expression, are not known. It appears that several critical steps in this intermediate series of regulatory reactions involve alterations in protein phosphorylation on serine and threonine residues. One subset of these responses are reflected by a rapid, insulin-induced increase in Ser/Thr phosphorylation of a number of proteins, which, in several instances, have been shown to result from the insulin activation of (Ser/Thr) specific protein kinases. It is proposed that such Ser/Thr protein kinases serve as intermediates to transmit, amplify and diversify the signal originated at the receptor tyrosine protein kinase. These Ser/Thr protein kinases appear to be representatives of an emerging class of insulin/growth factor-regulated Ser/Thr protein kinases, which may include as yet uncharacterized proto-oncogenes. The present proposal is devoted to the study of two insulin-activated protein kinases, whose best known substrate is the ribosomal 40S protein S6. The dominant insulin-activated S6 kinase in mammalian cells is a 70 kd polypeptide; we have shown that the catalytic activity of this kinase is stimulated through an insulin-induced Ser/Thr phosphorylation of the 70 kd polypeptide itself. The investigators have purified the 70 kd S6 kinase from rat liver and isolated a rat liver cDNA encoding the 70 kd kinase. By contrast, the dominant insulin-activated S6 kinase in Xenopus oocytes is an 85 kd polypeptide, which is also activated by an insulin-induced Ser/Thr phosphorylation. The investigators have cloned cDNAs encoding the rat liver homolog of the 85 kd S6 kinase, and using antiserum to the recombinant protein, shown that this enzyme is expressed and in mammalian cells, also regulated by insulin through Ser/Thr phosphorylation. The two S6 kinases are about 50 percent identical in amino acid sequence in their catalytic domains, but immunochemically distinct; moreover, whereas the 85 kd Xenopus S6 kinase can be activated in vitro by phosphorylation with a MAP-2 kinase, the 70 kd S6 kinase is not modified by MAP-2 kinase. The proposed studies are aimed at the detection, purification and characterization of the elements (? S6 kinase-kinases) required for insulin activation of these two, independently regulated S6 kinases, with the aim of reconstituting in vitro the entire series of reactions between the activated insulin receptor kinase and each S6 kinase. These studies may clarify the molecular mechanism of insulin action and provide new insights into the hormonal regulation of cell metabolism and growth. These results will have implications for the design of new pharmacologic in diabetes mellitus, and in understanding the disordered growth control which characterizes cancer.